Fluorescent turn-on chemosensors for detection of aluminum ion and azide

ABSTRACT

Two rhodamine derivatives, L1 and L2, bearing 2-methoxy-1-naphthaldehyde and 5-bromo-3-methoxy salicylaldehyde units were synthesized using microwave-assisted organic synthesis and used for reversible sequential fluorescence detection of aluminum ion (Al3+) and azide (N3−) in aqueous acetonitrile solution.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to rhodamine Schiff base compounds for thedetection of micromolar levels of Al³⁺ ions and azide (N₃ ⁻).

Description of the Background

Several approaches discuss detection of aluminum but not in conjunctionwith azide. U.S. Pat. No. 9,891,237 relies on a Schiff base for metalcation detection, but its sensor relies on a form of benzazole. U.S.Pat. No. 7,615,377 also uses ligands for detection of metal ions, and italso is based on fluorescence, but it does not apply the specificformula towards the same specific ligands. U.S. Pat. No. 7,906,320covers a fluorescence-based biosensor that can specifically detectmetals and also discusses quenchers that emit at specific wavelengthranges. U.S. Pat. No. 7,018,840 refers to fluorescent metal sensors,with rhodamine complexed with metal ions through ligand binding but doesnot list aluminum as one of the exemplary metal ions. U.S. Pat. No.5,567,619 detects for aluminum, among other elements/compounds and doesmention some other similar attributes, such as chelation and certaincolor indications, but it is overall more primitive in nature.

SUMMARY OF THE INVENTION

The present invention relates to sensor compounds (“sensors”) that aredeveloped from rhodamine derivatives that may be used for detecting thepresence of Al³⁺ and other metals.

Widespread use of aluminum in pharmaceuticals, cooking utensils,aluminum foil, vessels, and trays can result in the moderate increase inAl³⁺ concentration in food, and potentially damage the central nervoussystem in humans.

Novel and unobvious rhodamine Schiff base sensors L₁ and L₂ aredescribed herein that are able to detect micromolar levels of Al³⁺ ionsby the chelation-enhanced fluorescence (CHEF) process. Also of note,Al³⁺ complexes L₁-Al³⁺ and L₂-Al³⁺ behave as highly selectivechemosensors for N₃ ⁻ ions by quenching of the fluorescence inacetonitrile/water (CH₃CN/H₂O) medium at 25° C.

The rhodamine derivative sensors L₁ and L₂ bearing2-methoxy-1-naphthaldehyde and 5-bromo-3-methoxy salicylaldehyde unitswere designed and synthesized from the parent rhodamine B and aromaticaldehydes in a two-step Schiff base condensation, usingmicrowave-assisted organic synthesis (MAOS) and utilized towardssequential fluorescence detection of aluminum ion (Al³⁺) and azide (N₃⁻) in aqueous acetonitrile solution. Aluminum ion (Al³⁺) triggers theformation of highly fluorescent ring-open spirolactam.

A mixture of ethanol with compound 2 and 2-methoxy-1-naphthaldehyde orwith compound 2 and 5-bromo-3-methoxy salicylaldehyde was placed in areaction vial and then stirred before being placed in a biotagemicrowave reactor. The closed reaction vessel in both cases was rununder pressure and irradiated for 10 minutes. After cooling to roomtemperature, the resulting solid was filtered and washed three timeswith cold ethanol. After drying, the resulting sensor yield wasmeasured—the L₁ sensor yielded 92%, while the L₂ sensor yielded 88%.

Absorption spectra studies showed that on incremental addition of Al³⁺ions, the absorption intensity at 315 nm increased gradually and a newabsorption peak at 565 nm with a shoulder at 525 nm was generated byring opening with a visual color change from colorless to pink. Thewell-defined isosbestic points at 340 and 375 nm clearly indicates theformation of a new complex species between L₁ and Al³⁺. Absorptionspectra of sensors recorded with the continuous addition of Al³⁺ showeda continuous increase in the absorption at 565 nm and that was employedto calculate binding constants for L₁ and L₂ with Al³⁺ using theBenesi-Hildebrand method.

The plot of absorbance of L₁ at 565 nm as a function of mole fraction ofadded Al³⁺ metal ion reveals that these probes bind to the metal ion in1:1 stoichiometry. The fluorescence spectrum of sensors L₁ and L₂ showeda peak at 585 nm upon the addition of Al³⁺ corresponding to thedelocalization in the xanthenes moiety of rhodamine.

The fluorescence and colorimetric response of the L₁-Al³⁺ and L₂-Al³⁺complexes were quenched by the addition of N₃ ⁻, which extracted theAl³⁺ from the complexes and turned off the sensors, confirming that therecognition process is reversible. The recognition ability of thesensors was confirmed by fluorescence titration, Job's plot, 1H-NMRspectroscopy and density functional theory (DFT) calculations.

When L₁-Al³⁺ is used as the sensor for N₃ ⁻, high concentration ofCN-interference must be eliminated by using mesoporous carbon basedadsorbent. The addition of N₃ ⁻ to the L₁-Al³⁺ solution led to a changein color of the solutions from pink to colorless, which was observedwith the naked eye. The addition of N₃ ⁻ to the solution containingL₁-Al³⁺ complex resulted in the reversal of the Al³⁺ induced changes inthe emission band at 585 nm in the fluorescence emission spectra.

Gradual addition of N₃ ⁻ results in continuous decrease in the emissionintensity at 585 nm. Based on fluorescence data, the detection limit ofL₁-Al³⁺ or N₃ ⁻ was calculated as 12 μM. A similar finding was observedfor complex L₂-Al³⁺ towards N₃ ⁻ ions. The L₂-Al³⁺ system revealedremarkably selective fluorescence “off” behavior exclusively with N₃ ⁻.The limit of detection value for N₃ ⁻ ions was found at 18 μM. Theseresults show that L₁-Al³⁺ and L₂-Al³⁺ binds N₃ ⁻ ions with higherselectivity and the process is reversible.

Accordingly, there is provided according to an embodiment of theinvention, a compound having the formula:

There is further provided according to the invention a compound havingthe formula:

There is further provided according to the invention a method forsynthesizing the compound L₁, comprising

mixing a compound having the formula

with 2-methoxy-1-naphthaldehyde and ethanol, stirring a resultingmixture until homogenous, and irradiating the resulting mixture in amicrowave reactor.

There is further provided according to the invention a method forsynthesizing the compound method for synthesizing the compound L2,comprising

mixing a compound having the formula

with 5-bromo-3methoxy salicylaldehyde and ethanol, stirring a resultingmixture until homogenous, and irradiating the resulting mixture in amicrowave reactor.

There is further provided according to the invention a method fordetermining a presence of Al³⁺ in a sample, comprising: contacting thesample with a colorless solution comprising compound L₁ or L₂ andobserving whether the colorless solution turns pink in color, where achange in color of the solution to pink indicates the presence of Al³⁺in the sample. According to a further embodiment of the invention, thecolorless solution shows no absorption above 450 nm in UV-vis absorptionspectra, and an absorption peak above 525 nm indicates the presence ofAl³⁺ in the sample.

There is further provided according to the invention a method fordetermining a presence of N₃ ⁻ in a sample, comprising: contacting thesample with a pink solution comprising a compound having the formula

and observing whether the pink solution turns colorless, where a changein color of the solution from pink to colorless indicates the presenceof N₃ ⁻ in the sample. According to a further embodiment of theinvention, the pink solution shows an absorption peak above 525 nm inUV-vis absorption spectra, and no absorption above 450 nm indicates thepresence of N₃ ⁻ in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures and synthetic routes of L₁ and L₂.

FIG. 2 shows UV-vis spectra of L₁ (10 μM) with Al³⁺ (0-23 μM) inCH₃CN/H₂O (7:3 ν/ν) solution.

FIG. 3 shows an Absorbance Job's plot for determination of L₁-Al³⁺complex (10 μM) in CH₃CN/H₂O (7:3 ν/ν) solution.

FIG. 4 a is a fluorescence spectra of L₁ (10 μM) in CH₃CN/H₂O (7:3 ν/ν)solution (λ_(ex)=510 nm).

FIG. 4 b is a fluorescence spectra of L₂ (10 μM) with metal ions (10 μM)in CH₃CN/H₂O (7:3 ν/ν) solution (λ_(ex)=510 nm).

FIG. 5 is a fluorescence spectral titration of L₁ (10 μM) on theincremental addition of Al(NO₃)₃ (23 equivalents) (λ_(ex)=510 nm).

FIG. 6 shows the effect of pH on fluorescence intensity of sensors L₁and L₂ (10 μM).

FIG. 7 a shows a proposed binding mechanism of sensor L₁ towards Al³⁺ inthe presence and absence of azide (N₃ ⁻).

FIG. 7 b shows a proposed binding mechanism of sensor L₂ towards Al³⁺ inthe presence and absence of azide (N₃ ⁻).

FIG. 8 a shows the fluorescence spectra of L₁-Al³⁺ (1:1) with anions (10μM) (λ_(ex)=510 nm).

FIG. 8 b shows a fluorescence spectral titration of L₁-Al³⁺ (23equivalents of Al³⁺) on the incremental addition of N₃ ⁻ (up to 35equivalents) (λ_(ex)=510 nm).

FIG. 9 shows ¹H-NMR spectral changes of L₂ (8 mM) in DMSO-d₆ andtitrated with 0-1.0 equivalents of Al³⁺ in deuterated water.

FIG. 10 a shows optimized structures and energy correlation of theHOMO-LUMO gap between L₁ and L₁-Al³⁺ salt.

FIG. 10 b shows optimized structures and energy correlation of theHOMO-LUMO gap between L₂ and L₂-Al³⁺ complex.

DETAILED DESCRIPTION

Chemicals and Instruments

All reagents and solvents were purchased as analytical-grade and usedwithout further purification unless otherwise stated. Stock solutions ofmetal ions were prepared from their nitrate and chloride salts and anionspecies from their tetrabutylammonium salts. Distilled deionized waterwas used throughout the experiments. ¹H-NMR and ¹³C-NMR spectra wererecorded using an Avance 400 MHz spectrometer (Bruker Billerica,Karlsruhe, Germany) with tetramethylsilane (TMS) as internal standardand deuterated chloroform (CDCl₃) as solvent. NMR spectra were analyzedusing MestReNova software (version 10, Mestrela Research, FelicianoBarrera-Bajo, Spain). The IR spectrum was obtained using FT-IRspectrometer (Shimadzu, IRAffinity-1S, Columbia, Md., USA). Highresolution electrospray ionization mass spectrometry (ESI-MS) wasacquired with a Bruker Apex-Qe instrument. All UV-vis spectroscopyexperiments were recorded using a Cary UV/vis spectrophotometer 5000(Varian, Walnut Creek, Calif., USA). Fluorescence emission spectraexperiments were measured using a Cary 60 series spectrometer (Agilent,Walnut Creek, Calif., USA), with excitation and emission slit widths of5 nm and excitation wavelength at 510 nm. MAOS reactions were carriedout in a single mode Biotage Initiator 2.0 (Biotage, Uppsala, Sweden).

Microwave-Assisted Synthesis and Characterization of L₁ and L₂

Sensors L₁ and L₂ were synthesized from the parent rhodamine B andaromatic aldehydes (2-methoxy-1-naphthaldehyde and 5-bromo-3-methoxysalicylaldehyde) in a two-step Schiff base condensation using MAOSheating protocols, as shown in FIG. 1 . Compound 2 was synthesizedaccording to procedure reported in Xiang Y, Tong A, Jin P, Ju Y, Org.Lett 2006, 8, 2863.

Synthesis of Sensor L₁

Using microwave heating protocol: A mixture of compound 2 (105 mg, 0.230mmol), 2-methoxy-1-naphthaldehyde (41 mg, 0.220 mmol) and ethanol (2 ml)was placed in a 10 ml reaction vial. The resulting mixture was stirredto make it homogeneous and it was placed in the cavity of a biotagemicrowave reactor. The closed reaction vessel was run under pressure andirradiated for 10 min at 100° C. After cooling to room temperature, theresulting solid was filtered and washed three times with cold ethanol.After drying, the sensor L₁ was isolated to give in 92% yield. Meltingpoint: 244-246° C.; ¹H-NMR (CDCl₃), δ (ppm): 9.63 (1H, s, N═C—H); 8.77(1H, t, J=7.4 Hz, H—Ar), 7.74 (1H, d, J=8.4 Hz, H—Ar), 7.71 (1H, d,J=8.0 Hz, H—Ar), 7.63 (1H, d, J=7.7 Hz, H—Ar), 7.48-7.51 (2H, m, H—Ar),7.15-7.27 (2H, m, H—Ar), 7.12 (1H, d, J=8.4 Hz), 7.09 (1H, d, J=4.9 Hz),6.63 (2H, d, J=8.8 Hz), 6.44 (2H, d, J=2.2 Hz), 6.28 (2H, dd, J=8.8 Hz,2.6 Hz), 3.82 (3H, s, OCH₃), 3.31 (8H, q, J=6.9 Hz, NCH₂CH₃), 1.14 (12H,t, J=6.9 Hz, NCH₂CH₃). 13C-NMR (CDCl₃), δ (ppm): 164.6, 157.8, 153.4,151.7, 148.8, 147.6 (N═C—H), 137.6, 133.1, 131.9, 130.3, 129.2, 128.1,127.0, 126.7, 124.0, 123.2, 116.8, 112.9, 108.1, 107.9, 106.5, 104.6,79.9, 66.3 (spiro carbon), 56.7, 44.3 (NCH₂CH₃), 12.7 (NCH₂CH₃); HRMS(ESI): m/z calcd for C₄₀H₄₀N₄O₃: 625.3173; Found: 625.3176 [M+H]+.

Synthesis of Sensor L₂

Using microwave heating protocol: A mixture of compound 2 (100 mg, 0.220mmol), 5-bromo-3-methoxy salicylaldehyde (51 mg, 0.221 mmol) and ethanol(2 ml) was placed in a 10 ml reaction vial. The resulting mixture wasstirred to make it homogeneous and it was placed in the cavity of abiotage microwave reactor. The closed reaction vessel was run underpressure and irradiated for 10 min at 100° C. After cooling to roomtemperature, the resulting solid was filtered and washed three timeswith cold ethanol. After drying, the sensor L₂ was isolated to give in88% yield. ¹H-NMR (CDCl₃), δ (ppm): 11.11 (1H, s, —OH), 8.94 (1H, s,—CH═N), 7.96 (1H, t, J=6.6 Hz, —Ar), 7.49 (2H, m, —Ar), 6.86 (1H, d,J=6.6 Hz, —Ar), 7.50 (2H, s, —Ar), 6.51-6.43 (4H, m, —Ar), 6.25 (2H, d,J=7.5 Hz, —Ar), 3.82 (3H, s, —OCH₃), 3.31 (8H, q, NCH₂CH₃), 1.16 (12H,t, J=6.6 Hz, NCH₂CH₃) 13C-NMR (CDCl₃), δ (ppm): 163.6, 152.7, 148.5,146.6 (—CH═N), 138.5, 138.1, 137.7, 134.0, 128.9, 128.5, 127.5, 123.1,121.8, 121.3, 108.1, 108.0, 106.5, 104.8, 97.3, 80.9, 65.5 (spirocarbon), 56.1, 43.6 (NCH₂CH₃), 12.4 (NCH₂CH₃). HRMS (ESI): m/z calcd forC₃₆H₃₇BrN₄O₄: 669.2071; Found: 669.2076 [M+H]+.

General Procedure for the Spectroscopic Studies

All spectroscopic measurements were carried out in aqueous CH₃CN mediumat room temperature. Stock solutions of sensors L₁ and L₂ (1×10⁻³ M),selected salts of cations (1×10⁻³ M) and anions (1×10⁻⁴ M) were preparedin CH₃CN/H₂O. Thus, L₁-Al³⁺ and L₂-Al³⁺ solutions for N₃ ⁻ detectionwere prepared by addition of 1.0 equivalent of Al³⁺ to the solution ofboth L₁ and L₂ (20 μM) in Tris-HCl (10 mM, pH=7.2) buffer containingCH₃CN/H₂O (7:3, ν/ν) solution. The resulting solution was shaken wellbefore recording the spectra. Each fluorescence titration was repeatedat least thrice until consistent values were obtained. Jobs continuousvariation method was used for determining the binding stoichiometry ofthe complexation reaction. The association constant (K) was calculatedfrom absorbance studies by the linear Benesi-Hildebrand equation. Colorchanges in solution phase were observed visually under normal light andunder a hand-held UV lamp upon addition of various metal ions at roomtemperature.

Synthesis of Sensors L1 and L2

The synthesis of L₁ and L₂ were prepared in two steps with 92% and 88%overall yields respectively (FIG. 1 ). The results obtained indicatethat, unlike classical heating, MAOS results in higher yields, shorterreaction time, mild reaction condition, simple work-up procedure andbetter purity offer privilege over other methods where complexchromatographic techniques are required for purification of the targetcompounds. The structure of sensors was fully characterized by ¹H-NMR,¹³C-NMR, FT-IR and HRMS spectroscopy and all data are in accordance withthe proposed structure.

Absorption Spectra Studies

The metal ion sensing of L₁ and L₂ were first investigated by UV-visabsorption spectra. The colorless solutions were very weakly fluorescentand showed no absorption above 450 nm, properties which arecharacteristic of the predominant ring-closed spirolactam. Thepredominant spirolactam form was further confirmed by observation of thecharacteristic carbon resonance near 66 ppm for each of the sensors. TheUV-vis spectra of sensors were recorded in buffer at 25° C. and showedan absorption maximum at λ=315 nm, which may be attributed to theintramolecular π-π* charge transfer transition. On incremental additionof Al³⁺ ions, the absorption intensity at 315 nm increased gradually anda new absorption peak at 565 nm with a shoulder at 525 nm was generatedby ring opening with a visual color change from colorless to pink. Thewell-defined isosbestic points at 340 and 375 nm clearly indicates theformation of a new complex species between L₁ and Al³⁺ ion (FIG. 2 ).The absorption enhancement is high compared to other metal ions.Selectivity of L₁ was checked in the presence of other metal ions. Nosignificant change in the UV-vis spectrum was observed upon the additionof a 10 equivalent excess of other metal ions of interest: Na⁺, K⁺,Mg²⁺, Ca²⁺, Ni²⁺, Zn²⁺, Co²⁺, Hg²⁺, Pb²⁺, Fe2+, Fe³⁺, Cr²⁺ and Cu²⁺.Absorption spectra of sensors recorded with the continuous addition ofAl³⁺ showed a continuous increase in the absorption at 565 nm and thatwas employed to calculate binding constants for L₁ and L₂ with Al³⁺using the Benesi-Hildebrand method. The plot of absorbance of L₁ at 565nm as a function of mole fraction of added Al³⁺ metal ion reveals thatthese probes bind to the metal ion in 1:1 stoichiometry (FIG. 3 ). Thecomplex association constant (K) calculated through theBenesi-Hildebrand equation for Al³⁺ with L₁ and L₂ were found to be3.82×104 M⁻¹ and 2.41×104 M⁻¹, respectively.

Fluorescence Spectral Response of Sensors

To further explore the sensing behavior of L₁ for Al³⁺ ion, thefluorescence spectra of L₁ in CH₃CN with various metal ions wereexamined. The fluorescence spectra were obtained by excitation at 510nm, and both the excitation and emission slit were 5 nm. Thefluorescence intensity of L₁ upon the additions of metal ions in CH₃CNshowed a remarkable sensitivity and selectivity towards Al³⁺, eventhough there were relatively small effects with Cu²⁺ and Cr³⁺ (FIG. 4 a). There was a significant emission intensity enhancement with 1.0equivalent of Al³⁺ which indicates sensor L₁ is an excellent turn-onsensor for Al³⁺. This very high fluorescence enhancement is attributedto the formation of ring-open spirolactam in the presence of Al³⁺. Thisselectivity for Al³⁺ ions over all other ions is due to selectivechelate formation with L₁ to afford an L₁-Al³⁺ complex (See FIG. 7 a ).When illuminated with a hand-held UV lamp, the addition of Al³⁺ ions tosensor solution resulted in orange fluorescence emission from L₁solution (FIG. 5 ). The fluorescence profile of L₂ were very similar tothose for sensor L₁: again Al³⁺ registered the highest fluorescenceenhancement while other metal ions showed no significant enhancement(FIG. 4 b ). The fluorescence spectrum of sensors L₁ and L₂ showed apeak at 585 nm upon the addition of Al³⁺ corresponding to thedelocalization in the xanthenes moiety of rhodamine. It is assumed thatthe spirolactam form was opened upon the addition of Al³⁺ to sensors andmakes a highly delocalized π-conjugated stable complexes with Al³⁺through their active donor sites (e.g., N and O atoms) of receptor part,though other ions failed which basically indicates that the coordinatemoiety of L₁ and L₂ matches perfectly with Al³⁺ ions instead of theother ions. The detection limits of L₁ and L₂ for Al³⁺ ions wereestimated based on the fluorescence titration experiment as 32 μM and 47μM respectively. Furthermore, the effect of pH values on thefluorescence of L₁ and L₂ were also investigated in a pH range from 3 to10. FIG. 6 shows that for free L₁ and L₂ at pH<5, due to protonation ofthe open-ring of spirolactam, an obvious color change and fluorescenceturn-on appeared. Thus, all the optical measurements were performed inbuffer solution with a pH of 7 to keep the sensors in their ring closedform.

Detection of Azide (N₃ ⁻)

Investigation of the reversible binding nature of the sensors is shownin FIG. 8 and FIGS. 7 a and 7 b . Due to the high stability of AlN₃, theL₁-Al³⁺ and L₂-Al³⁺ complexes serve as a means to detect N₃ ⁻. FIG. 8 ashows the addition of 20 μM of ancions N₃ ⁻, CN⁻, ClO₄ ⁻, CH₃COO⁻, HSO₄⁻, H₂SO₄ ²⁻, SCN⁻, Cl⁻, I⁻, F⁻, and OH⁻ to L₁-Al³⁺ (1:1) of which N₃ ⁻alone quenches the fluorescence, with a slight effect for CN⁻,indicating high selectivity for N₃ ⁻. High concentration of CN⁻contamination is likely to mislead the fluorescent selectivity of N₃ ⁻.So, when L₁-Al³⁺ is used as the sensor for N₃ ⁻, high concentration ofCN⁻ interference must be eliminated by using mesoporous carbon basedadsorbent. The addition of N₃ ⁻ to the L₁-Al³⁺ solution led to a changein color of the solutions from pink to colorless, which was observedwith the naked eye. The addition of N₃ ⁻ to the solution containingL₁-Al³⁺ complex resulted in the reversal of the Al³⁺ induced changes inthe emission band at 585 nm in the fluorescence emission spectra.Gradual addition of N₃ ⁻ results in continuous decrease in the emissionintensity at 585 nm (FIG. 8 b ). Based on fluorescence data, thedetection limit of L₁-Al³⁺ for N₃ ⁻ was calculated as 12 μM. A similarfinding was observed for complex L₂-Al³⁺ towards N₃ ⁻ ions. The L₂-Al³⁺system revealed remarkably selective fluorescence “off” behaviorexclusively with N₃ ⁻. The limit of detection value for N₃ ⁻ ions wasfound at 18 μM. These results show that L₁-Al³⁺ and L₂-Al³⁺ bind N₃ ⁻ions with higher selectivity and that the process is reversible. Theexpected binding mechanism of sensors with Al³⁺ in the presence andabsence of azide (N₃ ⁻) is shown in FIGS. 7 a and 7 b.

FT-IR and ¹H-NMR Study for Elucidation of Coordination Mechanism BetweenSensors and Al³⁺

To elucidate the coordination mechanism of L₁-Al³⁺ and L₂-Al³⁺complexes, the FT-IR spectrum of L₁ and L₂ were conducted in the absenceand presence of Al³⁺ ion. The characteristic peak of the amide carbonylγ_((C═O)) shifted from 1680 cm⁻¹ to 1614 cm⁻¹ in the presence of Al³⁺,indicating that carbonyl O atoms of the L₁ and L₂ are involved in thecoordination of Al³⁺. ¹H-NMR was also performed by adding Al³⁺ todeuterated dimethyl sulfoxide (DMSO-d₆) solution of L₂ as shown in FIG.9 . The L₂-Al³⁺ complexes were prepared by the additions of 0.25, 0.5and 1.0 equivalent AlCl₃·6H₂O to the DMSO solution of L₂. The peaksobserved at δ 10.10 and δ 9.07 are attributable to the phenolic OH andthe imine proton (—CH═N—) in L₂. Addition of 1 equivalent of Al³⁺resulted in the disappearance of the hydroxyl proton indicating thebinding of Al³⁺ ion through the phenoxide interaction. Further, smallunfilled-shifts from 9.07 to 9.00 ppm and shortening of imine protonswere observed because of the complex formation between nitrogen atomsand Al³⁺. The formation of the L₂-Al³⁺ complex through normal ringopening was confirmed by performing the ¹³C-NMR experiment with L₂ inthe absence and presence of Al³⁺ ions, from which it was observed thatthe signal at δ=66 ppm attributable to the tertiary carbon of thespirolactam ring in L₂ was absent from the spectrum of L₂-Al³⁺ complex.Therefore, it is understood that the O atom of phenolic OH, N atom ofimine and O atom of spiro ring coordinate to Al³⁺ as shown in FIGS. 7 aand 7 b.

Geometry Optimization

To better understand the nature of the coordination of Al³⁺ withsensors, theoretical calculations on structures L₁, L₂, L₁-Al³⁺ andL₂-Al³⁺ were carried out using Spartan '16 software. Density functionaltheory (DFT), employing the B3LYP functional and the 6-31G* basis setwas used to obtain gas phase, optimized geometries of these structures.The optimized structures of L₁, L₂ and their respective Al-complexes aredepicted in FIGS. 10 a and 10 b. L₁ and L₂ can undergo rotation ofapproximately 180° about the N—N bond, producing two prominent cis andtrans conformations. For both L₁ and L₂, the trans conformation is moreenergetically stable than the respective cis one by approximately 11.3kJ mol⁻¹, owing to anti-arrangement of the methoxy (—OMe) group and thexanthene moiety in trans L₁ and to the anti-arrangement of the hydroxyl(—OH) group and the xanthene moiety in trans L₂. Additionally, in transL₁ the energy gap between the highest occupied molecular orbital (HOMO)(−4.81 eV) and the lowest unoccupied molecular orbital (LUMO) (−1.34 eV)is 3.47 eV, and in cis L₁ the gap, HOMO (−5.03 eV) and LUMO (−1.35 eV),is 3.68 eV. In trans L₂, the energy gap, HOMO (−4.86 eV) and LUMO (−1.29eV) is 3.57 eV, and in cis L₂ the energy gap, HOMO (−5.05 eV) and LUMO(−1.22 eV) is 3.83 eV, suggesting that trans L₁ and trans L₂ are themajor equilibrium conformations available stereochemically for directAl³⁺ coordination. Also, in trans L₁, the electron density isdelocalized over the entire xanthene moiety with some found on thespirolactam ring as well as on the imine and the ortho-methoxynaphthalene moieties (FIG. 10 a ). In cis L₁, the electron density ismainly localized on half of the xanthene moiety. In both trans L₂ andcis L₂, the electron density is mainly located over the entire xanthenemoiety with some found on the lactam ring nitrogen of both. Moreover,some electron density is also found on the carbonyl oxygen in trans L₂but not on the carbonyl oxygen in cis L₂ (FIG. 10 b ).

Density functional calculations of molecular interactions of trans-L₁and trans-L₂ with aqueous aluminum (Al³⁺) nitrate solution revealed thatboth sensors are energetically stabilized on binding with Al³⁺ ions. Forinstance, upon formation of L₁-Al³⁺ salt complex, the HOMO-LUMO energygap in trans-L₁ (ΔE=3.47 eV) decreased to ΔE=2.40 eV, and upon formationof L₂-Al3+ complex, the HOMO-LUMO energy gap in trans-L₂ (ΔE=3.57 eV)decreased to 2.22 eV. In L₁-Al³⁺ salt complex, formulated as [Al (L₁)NO₃)₂(H₂O)₂] [NO₃], HOMO is primarily delocalized over the methoxynaphthalene moiety, while LUMO is primarily delocalized over thexanthene moiety. In L₂-Al³⁺ complex, formulated as Al (L₂) (NO₃)₂(H₂O),HOMO is found over the tricyclic structure about Al³⁺ while LUMO isdelocalized over the xanthene moiety (FIGS. 10 a and 10 b ).

Vertical electronic excitations of optimized B3LYP/6-31G* trans-L₁,trans-L₂ and their respective complexes were computed usingtime-dependent-density functional theory (TD-DFT) Spartan '16 softwarecalculations, formalized in water and using a conductor-like polarizablecontinuum model (CPCM). In the TD-DFT UV-vis spectrum of trans-L₁, anabsorption band at λ=379.24 nm with a vertical excitation energy of3.2693 eV and corresponding to HOMO-2→LUMO excitation (oscillatorstrength=0.4632) dominates. While in the TD-DFT UV-vis spectrum oftrans-L₁-Al³⁺ salt complex, an absorption band at λ=422.57 nm dominates,corresponding to HOMO→LUMO excitation (vertical excitation energy=2.9341eV and oscillator strength=1.0951). In the case of trans-L₂, anabsorption band at λ=344.32 nm dominates, corresponding to HOMO-2→LUMOexcitation with a vertical excitation energy of 3.6008 eV and anoscillator strength=0.3152. For trans-L₂-Al³⁺ complex, an absorptionband at λ=456.19 nm dominates, corresponding to HOMO-1→LUMO andHOMO→LUMO excitations with a vertical excitation energy of 2.7178 eV andan oscillator strength=0.7824.

CONCLUSION

We have developed reversible fluorescent sensors L₁ and L₂ for theselective and sensitive sequential detections of Al³⁺ and N₃ ⁻ via thefluorescence spectral changes. Upon binding to Al³⁺, obvious detectablechange in fluorescence was observed due to the CHEF effect. The in situprepared L₁-Al³⁺ and L₂-Al³⁺ complexes were used to detect N₃ ⁻ via themetal-displacement approach which displayed an excellent selectivity andsensitivity towards N₃ ⁻. Thus, upon the addition of N₃ ⁻ to complexes,the intensity of the 585 nm band decreases, indicating release of L₁ andL₂ from the aluminum complexes. Stoichiometry and binding mechanisms forboth sensors are well characterized and established by the respectivespectroscopic techniques. These results clearly demonstrate that L₁ andL₂ sensors described herein will be useful for the analysis of Al³⁺ andN₃ ⁻ in environmental samples and biological studies.

1. A compound having the formula:


2. A compound having the formula:


3. A method for synthesizing the compound L₁ of claim 1, comprisingmixing a compound having the formula

with 2-methoxy-1-naphthaldehyde and ethanol, stirring a resultingmixture until homogenous, and irradiating the resulting mixture in amicrowave reactor.
 4. A method for synthesizing the compound L₂ of claim1, comprising mixing a compound having the formula

with 5-bromo-3methoxy salicylaldehyde and ethanol, stirring a resultingmixture until homogenous, and irradiating the resulting mixture in amicrowave reactor.
 5. A method for determining a presence of Al³⁺ in asample, comprising: contacting the sample with a colorless solutioncomprising a compound according to claim 1 and observing whether thecolorless solution turns pink in color, where a change in color of thesolution to pink indicates the presence of Al³⁺ in the sample.
 6. Amethod for determining a presence of N₃ ⁻ in a sample, comprising:contacting the sample with a pink solution comprising a compoundaccording to claim 2 and observing whether the pink solution turnscolorless, where a change in color of the solution from pink tocolorless indicates the presence of N₃ ⁻ in the sample.
 7. A methodaccording to claim 5 wherein the colorless solution shows no absorptionabove 450 nm in UV-vis absorption spectra, and wherein an absorptionpeak above 525 nm indicates the presence of Al³⁺ in the sample.
 8. Amethod according to claim 6 wherein the pink solution shows anabsorption peak above 525 nm in UV-vis absorption spectra, and whereinno absorption above 450 nm indicates the presence of N₃ ⁻ in the sample.